DETECTION KIT FOR QUATERNARY AMMONIUM COMPOUND HAVING y-CARBOXYL GROUP

ABSTRACT

The invention discloses a detection kit for a quaternary ammonium compound having a γ-carboxyl group. The kit includes a fluorescent compound having a fluorophore. The fluorophore of the fluorescent compound is used for forming a derivative with the γ-carboxyl group of the quaternary ammonium compound by an SN2 nucleophilic substitution reaction. The kit further includes a polar aprotic solvent used for accelerating the SN2 nucleophilic substitution reaction.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 15/616,734 filed on Jun. 7, 2017. The application claims thebenefit both of U.S. provisional application No. 62/400,120, filed onSep. 27, 2016, and Taiwan application serial No. 106103831, filed Feb.6, 2017, the subject matter of which is incorporated herein byreference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention generally relates to a detection method, adiagnostic method utilizing the detection method and a detection kitutilizing the detection method and, more particularly, to a detectionmethod for a quaternary ammonium compound having a γ-carboxyl group, adiagnostic method for carnitine deficiency and a detection kit for thequaternary ammonium compound having the γ-carboxyl group.

2. Description of the Related Art

Quaternary ammonium compounds having a γ-carboxyl group, such asL-carnitine and the derivatives, acyl-L-carnitine and acetyl-L-carnitineare important molecules for human bodies. However, those compounds arecharacterized in low molecular weight, but also have high polarity dueto the γ-carboxyl group and the ammonium group; and therefore beingdifficult to be extracted. Moreover, the quaternary ammonium compoundslack fluorescent group such as fluorophore. Therefore, conventionaldetection methods are not significant for detecting the quaternaryammonium compounds.

SUMMARY OF THE INVENTION

It is therefore the objective of this invention to provide a detectionmethod for a quaternary ammonium compound having a γ-carboxyl group,assuring the quaternary ammonium compound to be tagged by a fluorophorewhich is adapted to be detected by a fluorescence spectrometry.

It is another objective of this invention to provide a diagnostic methodfor carnitine deficiency, by detecting L-carnitine amount in a samplefrom a suspected patient by said detection method to evaluate whetherthe suspected patient suffers from carnitine deficiency.

It is yet another objective of this invention to provide a detection kitfor the quaternary ammonium compound having the γ-carboxyl group, whichis adapted to detect whether said quaternary ammonium compound exists ina sample.

One embodiment of the invention discloses a detection method for aquaternary ammonium compound having a γ-carboxyl group, comprising:mixing a sample comprising the quaternary ammonium compound having theγ-carboxyl group, a fluorescent compound having a fluorophore and apolar aprotic solvent to form a reaction solution; incubating thereaction solution at 80-120° C. for 1-15 minutes, assuring an S_(N)2nucleophilic substitution reaction occurs between the fluorophore of thefluorescent compound and the γ-carboxyl group of the quaternary ammoniumcompound, to obtain a derivation solution including a derivative whichis a quaternary ammonium compound with the γ-carboxyl group substitutedwith the fluorophore of the fluorescent compound; and detecting thederivative in the derivation solution as an analytic solution. Thesample can be selected from a pharmaceutical sample, a cosmetic sample,a food sample or a biological sample derived from a mammal.

In a preferred form shown, the fluorescent compound having thefluorophore can be selected from 4-bromomethylbiphenyl,4-bromoethylbiphenyl, 4-bromomethyl-2′-cyanobiphenyl,(2-biphenyl)diazomethane, 4-aminobiphenyl, biphenyl-4-yl-hydrazinehydrochloride, 4-phenylphenol or 4-biphenylyl trifluoromethanesulfonate.

In a preferred form shown, the polar aprotic solvent can be selected asacetonitrile.

In a preferred form shown, a base is added to the reaction solution,followed by assuring the S_(N)2 nucleophilic substitution reactionoccurs in the reaction solution dissolving the base to obtain thederivation solution.

In a preferred form shown, the base is selected from potassiumhydroxide, potassium carbonate or potassium hydrogen carbonate.

In a preferred form shown, reverse-phase liquid chromatography,fluorescence spectrometry or mass spectrometry is used to detect thederivative in the analytic solution.

In a preferred form shown, an extractant is added to the derivationsolution to extract the derivative in the derivation solution, obtainingthe analytic solution. The polarity of the extractant is higher than thepolar aprotic solvent.

In a preferred form shown, at the time of adding the extractant to thederivation solution, adding a cosolvent to the derivation solution,assuring the extractant and the cosolvent form a water-in-oil emulsionwhich disperses in the derivation solution, followed by extracting thederivative in the derivation solution by the extractant.

In a preferred form shown, the extractant is selected from a deionizedwater, an aqueous ammonium acetate solution, an aqueous ammoniumchloride solution, an aqueous sodium chloride solution or an aqueousammonium hydrogen carbonate solution. The cosolvent is selected astoluene.

Another embodiment of the invention discloses a diagnostic method forcarnitine deficiency, comprising: obtaining a suspected sample from asuspected patient; detecting an amount of carnitine derivatives in thesuspected sample ex vivo by said detection method to obtain a detectionvalue; and comparing the detection value of the suspected sample with areference value. The detection value is lower than the reference valueindicates that the suspected patient suffers from carnitine deficiency.The suspected sample is a whole blood sample, a serum sample, a plasmasample or a urine sample obtained from the suspected patient.

In another preferred form shown, the detection value is obtained bydetecting an L-carnitine amount of the suspected sample from thesuspected patient. The reference value is obtained by detecting anL-carnitine amount of a sample from a healthy subject.

In another preferred form shown, a L-carnitine amount and an acetylL-carnitine amount of the sample from the suspected patient aredetected, respectively, and the detection value is the ratio between theL-carnitine amount and the acetyl L-carnitine amount of the suspectedsample from the suspected patient. A L-carnitine amount and an acetylL-carnitine amount of the sample from a healthy subject are detected,respectively, and the reference value is the ratio between theL-carnitine amount and the acetyl L-carnitine amount of the sample fromthe healthy subject

The other embodiment of the invention discloses a detection kit for aquaternary ammonium compound having a γ-carboxyl group, comprising: afluorescent compound having the fluorophore and a polar aprotic solvent.The fluorophore of the fluorescent compound is used for forming aderivative with the γ-carboxyl group of the quaternary ammonium compoundvia an S_(N)2 nucleophilic substitution reaction. The polar aproticsolvent is used for accelerating the S_(N)2 nucleophilic substitutionreaction.

In the other preferred form shown, the fluorescent compound is selectedfrom 4-bromomethylbiphenyl, 4-bromoethylbiphenyl,4-bromomethyl-2′-cyanobiphenyl, (2-biphenyl)diazomethane,4-aminobiphenyl, biphenyl-4-yl-hydrazine hydrochloride, 4-phenylphenolor 4-biphenylyl trifluoromethanesulfonate.

In the other preferred form shown, the polar aprotic solvent isacetonitrile.

In the other preferred form shown, the detection kit comprises: a base.The base is a basic compound dissolvable in the polar aprotic solventand used for providing a basic environment for the S_(N)2 nucleophilicsubstitution reaction.

In the other preferred form shown, the base is selected from potassiumhydroxide, potassium carbonate or potassium bicarbonate.

In the other preferred form shown, the detection kit comprises: anextractant. The extractant has a polarity higher than the polar aproticsolvent thereof and is used for extracting the derivative.

In the other preferred form shown, the detection kit comprises: acosolvent. The cosolvent is used for forming a water-in-oil emulsionwith the extractant.

In the other preferred form shown, the extractant is selected from adeionized water, an aqueous ammonium acetate solution, an aqueousammonium chloride solution, an aqueous sodium chloride solution or anaqueous ammonium hydrogen carbonate solution, while the cosolvent istoluene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a depicts a chemical reaction of the S_(N)2 nucleophilicsubstitution reaction between L-carnitine and 4-bromomethylbiphenyl(Br-MBP).

FIG. 1b depicts a chemical reaction of the S_(N)2 nucleophilicsubstitution reaction between acetyl-L-carnitine and4-bromomethylbiphenyl (Br-MBP).

FIG. 2 depicts a bar chart showing the peak area ratio of groups A11-A16and A21-A26.

FIG. 3 depicts a bar chart showing the peak area ratio of groups B11-B16and B21-B26.

FIG. 4 depicts a bar chart showing the peak area ratio of groups C10-C13and C20-C23.

FIG. 5 depicts a bar chart showing the peak area ratio of groups C11 andC21 which are incubated at 80° C.

FIG. 6 depicts a bar chart showing the peak area ratio of groups C11 andC21 which are incubated at 90° C.

FIG. 7 depicts a bar chart showing the peak area ratio of groups C11 andC21 which are incubated at 100° C.

FIG. 8 depicts a bar chart showing the peak area ratio of groups E11-E16and E21-E26.

FIG. 9 depicts a bar chart showing the peak area ratio of groups F11-F16and F21-F26.

FIG. 10 depicts a bar chart showing the peak area ratio of groupsG10-G14 and G20-G24.

FIG. 11 depicts a bar chart showing the peak area ratio of groupsH10-H14 and H20-H24.

DETAILED DESCRIPTION OF THE INVENTION

The “quaternary ammonium compound having a γ-carboxyl group” accordingto the present application indicates a quaternary ammonium compound witha carbon chain which has a carboxyl group (—C(═O)OH) on thegamma-position carbon (Cγ). In an example, the quaternary ammoniumcompound can be butyrobetaine, L-carnitine, acyl-L-carnitine species,carnitine tartrate, carnitine fumarate or carnitine citrate, etc.Moreover, the acyl-L-carnitine species includes, but not limited to,acetyl-L-carnitine, propionyl-L-carnitine, butyryl-L-carnitine,isovaleryl-L-carnitine, hexanoyl-L-carnitine, octanoyl-L-carnitine,decanoyl-L-carnitine, lauroyl-L-carnitine, myristoyl-L-carnitine,palmitoyl-L-carnitine stearoyl-L-carnitine, etc., which can beappreciated by a person having ordinary skill in the art.

According to an embodiment of the present application, a detection kitfor the quaternary ammonium compound can include a fluorescent compoundwith a fluorophore, and polar aprotic solvent. The detection kit can beadapted to detect whether the quaternary ammonium compound exists in asample.

The sample can be selected from a pharmaceutical sample, a cosmeticsample, a food sample or a biological sample derived from a mammal.Specifically, an appropriate pretreat step, such as centrifugation ordrying, can be performed before detecting the sample using the detectionkit according to the type of the sample, which can be appreciated by aperson having ordinary skill in the art.

More specifically, the sample, the fluorescent compound and the polaraprotic solvent can be mixed to form a reaction solution, assuring anS_(N)2 nucleophilic substitution reaction occurs between the fluorophoreof the fluorescent compound and the γ-carboxyl group of the quaternaryammonium compound to form a derivative. The derivative, afluorescent-labeled quaternary ammonium compound, is a quaternaryammonium compound with the fluorophore of the fluorescent compound beingsubstituted on the γ-carboxyl group. The reaction solution including thederivative is so-called a “derivation solution” in the followingdescription. So that, the derivation solution can be used as an analyticsolution, and the derivative in the analytic solution can be detected,evaluating whether the sample includes the quaternary ammonium compoundhaving the γ-carboxyl group.

The fluorescent compound includes the fluorophore and a leaving group.In the S_(N)2 nucleophilic substitution reaction, the γ-carboxyl groupof the quaternary ammonium compound deprotonates in a natural or a basicenvironment, attacking the fluorescent compound and releasing theleaving group from the fluorescent compound. Thus, the quaternaryammonium compound can form the derivative together with the fluorophoreof the fluorescent compound. As an example, the fluorescent compound canbe selected from, but not limited to, 4-bromomethylbiphenyl (Br-MBP),4-bromoethylbiphenyl, 4-bromomethyl-2′-cyanobiphenyl,(2-biphenyl)diazomethane, 4-aminobiphenyl, biphenyl-4-yl-hydrazinehydrochloride, 4-phenylphenol or 4-biphenylyl trifluoromethanesulfonate.The exemplified S_(N)2 nucleophilic substitution reactions, in whichL-carnitine and acyl-L-carnitine respectively react with Br-MBP areshown in FIGS. 1a and 1 b.

The polar aprotic solvent is used for accelerating the S_(N)2nucleophilic substitution reaction. As an example, the polar aproticsolvent can be acetonitrile (ACN).

It is worthy to note that when forming the reaction solution, thesample, the fluorescent compound and the polar aprotic solvent can bemixed at the same time. Alternatively, the sample and the fluorescentcompound can be respectively dissolved in the polar aprotic solvent,forming a sample solution and a fluorescent compound solution, so thatthe concentration and the amount of the sample, the fluorescent compoundand the polar aprotic solvent can be easily calculated. In thisembodiment, the sample solution (2 μM) is formed by dissolving thesample in the polar aprotic solvent, and the fluorescent compoundsolution (2-35 mM) is formed by dissolving the fluorescent compound inthe polar aprotic solvent. The reaction solution is then formed bymixing the sample solution (20 μL) and the fluorescent compound solution(5 μL).

Besides, the detection kit can further include a base. The base is ableto promote the deprotonation of the γ-carboxyl group of the quaternaryammonium compound, improving the efficiency of the S_(N)2 nucleophilicsubstitution reaction. The base is a basic compound dissolvable in thepolar aprotic solvent, such as potassium hydroxide (KOH), potassiumcarbonate (K₂CO₃) or potassium hydrogen carbonate (NaHCO₃). Preferably,potassium hydroxide can be used as the base. In addition, the base canbe mixed with the polar aprotic solvent to form a saturated basicsolution which provide an appropriate basic environment and improve theefficiency of the S_(N)2 nucleophilic substitution.

Moreover, the base, the sample, the fluorescent compound and the polaraprotic solvent can be mixed at the same time. Alternatively, the basecan be dissolved in the polar aprotic solvent to form a basic solution.The basic solution can then be mixed with the sample solution and thefluorescent compound solution. In this embodiment, the saturated basicsolution is formed by dissolving the base in the polar aprotic solvent.The reaction solution is then formed by mixing the sample solution (20μL), the fluorescent compound solution (5 μL) and the saturated basicsolution (2 μL).

In order to accelerate the S_(N)2 nucleophilic substitution reaction,the worker can further apply thermal energy to the reaction solution. Inthis embodiment, the reaction solution is incubated at 80-120° C. for1-15 minutes and the derivation solution is then obtained. For example,the reaction solution can be heated at 80° C. for 9 minutes or at90-100° C. for 7 minutes to quickly complete said S_(N)2 nucleophilicsubstitution reaction. Furthermore, the heating time can be adjustedaccording to the status of the container containing the reactionsolution, preventing the polar aprotic solvent from evaporation.

The derivation solution obtained via the S_(N)2 nucleophilicsubstitution reaction can be used as the analytic solution. The analyticsolution can be analyzed by reverse-phase liquid chromatography,fluorescence spectrometry or mass spectrometry. As an example, thereverse-phase liquid chromatography can be performed by a high pressureliquid chromatography system such as a narrow-bore liquid chromatographysystem or a nano liquid chromatography system. Specifically, beforebeing analyzed by the reverse-phase liquid chromatography, theconcentration or the polarity of the analytic solution can be adjusted,followed by mixing with an internal control. Then, the analytic solutionwith the internal control is loaded in the narrow-bore liquidchromatography system or the nano liquid chromatography system. In thisembodiment, the analytic solution is mixed with methanol and9-aminoacridine (as the internal standard), then analyzed by thenarrow-bore liquid chromatography system. Moreover, the mobile phaseused for gradient elution includes methanol and a formic acid solution(FA_((aq))) containing 0.2% formic acid.

The mass spectrometry can be performed by a m/z detector, which has alow detection limitation and a high sensitivity. In addition, thederivative (i.e., the fluorophore-labeled quaternary ammonium compound)in the analytic solution has a fluorophore substituting via the S_(N)2nucleophilic substitution reaction thus can be analyzed by thefluorescence spectrometry, using an instrument named the fluorescencedetector. When the derivative is analyzed by the fluorescencespectrometry, the wavelength of the highest excitation intensity is at255 mm, and the wavelength of the highest emission intensity is at 317nm.

Moreover, the detection kit can further include an extractant. Theextractant has a polarity higher than a polarity of the polar aproticsolvent and can be used for extracting the derivative in the derivationsolution to obtain an extract. The extract can also be used as theanalytic solution, and analyzed by the liquid chromatography, thefluorescence spectrometry or the mass spectrometry according to demand.

The extractant can be selected from deionized water or an aqueous saltsolution. The aqueous salt solution can be selected from an aqueousammonium acetate solution (NH₄OAc_((aq))), an aqueous ammonium chloridesolution (NH₄Cl_((aq))), an aqueous sodium chloride solution(NaCl_((aq))) or an aqueous ammonium hydrogen carbonate solution(NH₄HCO_(3 (aq))). Preferably, the extractant is the aqueous saltsolution which dissociates and forms ions when being mixed with thederivation solution, facilitating the extraction of the derivative. Theaqueous salt solution has a preferable concentration being 1 M.Moreover, under the circumstances that the sample is selected as aplasma sample, the extractant is preferably an aqueous ammonium solutionin the concentration of 1 M.

The detection kit can further includes a cosolvent. The cosolvent is anorganic solvent having a density lower than 1 g/cm³ and cannot dissolvein the extractant; and therefore, after the extractant and the cosolventare added to the derivation solution, the derivation solution, theextractant and the cosolvent can form a cloudy mixture by vortexing.Specifically, the extractant and the cosolvent form a water-in-oilemulsion dispersing in the derivation solution in the form of droplets,increasing the contacting area between the extractant and the derivationsolution. With such performance, the effect of the extractant extractingthe derivative from the derivation solution is improved. As an example,the cosolvent can be toluene.

Moreover, the extract can be collected after the extraction. As anexample, the mixture containing the derivation solution, the extractantand the cosolvent can be separated into different layers bycentrifugation, and the extract is obtained by collecting the aqueousphase which distributes in the lower layer.

Besides, in this embodiment, at the time of mixing the derivationsolution (including acetonitrile as the polar aprotic solvent), theextractant and the cosolvent, acetonitrile can facilitate the extractantdispersing in the derivation solution; and therefore, the efficiency ofthe extraction can be improved.

The detection kit is able to be adapted to trigger the formation of thederivative (i.e., the fluorophore-labeled quaternary ammonium compound)via the S_(N)2 nucleophilic substitution reaction, the derivative cansubsequently be analyzed by the reverse-phase liquid chromatography, thefluorescence spectrometry or the mass spectrometry. Therefore, thedetection kit can be adapted to a detection method for the quaternaryammonium compound having the γ-carboxyl group. Specifically, thedetection method includes: mixing the sample, the fluorescent compoundand the polar aprotic solvent to form the reaction solution; incubatingthe reaction solution at 80-120° C. for 1-15 minutes, assuring theS_(N)2 nucleophilic substitution reaction occurs between the fluorophoreof the fluorescent compound and the γ-carboxyl group of the quaternaryammonium compound, to obtain a derivation solution comprising thederivative; and analyzing the derivative in the derivation solution asthe analytic solution.

Furthermore, the detection method according to an embodiment of thepresent application includes adding the extractant in the derivationsolution to extract the derivative from the derivation solution toobtain the analytic solution.

It is worthy to note that the detection kit can be used for detectingthe quaternary ammonium compound in the biological sample derived from amammal; and therefore, the detection kit and the detection method can beadapted to measure the amount of carnitine derivatives in a suspectedpatient, evaluating whether the suspected patient suffers from carnitinedeficiency. Specifically, a suspected sample is first obtained from thesuspected patient and a control sample is obtained from a healthysubject. Both the suspected sample and the control sample can beselected as whole blood samples, serum samples, plasma samples or urinesamples. The amounts of carnitine derivatives in the suspected sampleand the control sample are measured by the detection method ex vivo toobtain a detection value and a reference value, respectively. Finally,the suspected patient is considered as a patient suffering fromcarnitine deficiency when the detection value is lower than thereference value.

For example, Inoue F et al. and Peng M et al. report that the amount ofL-carnitine in a plasma sample obtained from healthy adults is about17.51-66.14 μM (J Chromatogr B Biomed Sci Appl. 1999 Aug. 6; 731(1):83-8.; J Chromatogr A. 2013 Dec. 6; 1319: 97-106.); and therefore, thedetection value can be the amount of L-carnitine of the suspectedpatient, and said reference value can be the amount of L-carnitine ofthe healthy subject. Specifically, when said detection value is theamount of L-carnitine in the plasma sample obtained from the suspectedpatient, said reference value is 66.14 μM. In addition, Hothi D K et al.and Flanagen J L et al. report that the ratio of acetyl-L-carnitine toL-carnitine (i.e., acetyl-L-carnitine/L-carnitine) in the plasma samplecan be considered as a health index, and a subject is considered to besuffered from carnitine deficiency when the ratio of acetyl-L-carnitineto L-carnitine in the plasma sample obtained from the subject is higherthan 0.25 (Nephrol Dial Transplant. 2006 September; 21(9): 2637-2641.)Accordingly, said detection value can be the ratio of L-carnitine toacetyl-L-carnitine of the suspected patient, and said reference valuecan be the ratio of L-carnitine to acetyl-L-carnitine of the healthysubject. Specifically, when said detection value is the ratio ofL-carnitine to acetyl-L-carnitine in the plasma sample obtained from thesuspected patient, said reference value is 0.25.

To evaluate the detection kit and the detection method can be used todetect the quaternary ammonium compound, the following trials areperformed.

Trial (A): The Effect of the Concentration of the Fluorescent compoundon the S_(N)2 nucleophilic substitution reaction.

The quaternary ammonium compound (i.e., L-carnitine for groups A11-A16or acetyl-L-carnitine for groups A21-A26) is dissolved in the polaraprotic solvent (i.e., ACN) and mixed with the fluorescent compound(i.e., Br-MBP) to form the reaction solution in accordance with theconcentrations indicated in TABLE 1. The reaction solution listed inTABLE 1 is incubated at 80-120° C. for 1-15 minutes to obtain thederivation solution. The derivative in the derivation solution isextracted by the extractant (i.e., deionized water) in the presence ofthe cosolvent (i.e., toluene), the aqueous phase is then collected andmixed with 9-aminoacridine as the internal control to obtain theanalytic solution. Finally, the derivative in the analytic solution isdetected by the narrow-bore LC-FLD system. The peak area ratio shown inFIG. 2 is calculated as the peak area of the derivative extracted by theextractant in the presence of the cosolvent divided by the peak area ofthe internal standard.

TABLE 1 Reaction solution Quaternary ammonium Group compound Fluorescentcompound A11 L-carnitine (1.6 μM) Br-MBP (0.4 mM) A12 Br-MBP (1 mM) A13Br-MBP (2 mM) A14 Br-MBP (3 mM) A15 Br-MBP (5 mM) A16 Br-MBP (7 mM) A21Acetyl-L-carnitine (1.6 μM) Br-MBP (0.4 mM) A22 Br-MBP (1 mM) A23 Br-MBP(2 mM) A24 Br-MBP (3 mM) A25 Br-MBP (5 mM) A26 Br-MBP (7 mM)

Referring to FIG. 2, in all of groups A11-A16 and A21-A26, the S_(N)2nucleophilic substitution reaction occurs, and the derivative can bedetected by the narrow-bore LC-FLD system. Moreover, in groups A11-A14and A21-A24, as the concentration of Br-MBP increases, the reactivity ofthe S_(N)2 nucleophilic substitution reaction increases. However, ingroups A15-A16 and A25-A26, even the concentration of Br-MBP is over 3mM, the reactivity of the S_(N)2 nucleophilic substitution reactionincreases no more. That is, the reaction solution preferably includes 3mM of the fluorescent compound in this embodiment.

Trial (B): The Effect of the Amount of the Fluorescent Compound on theS_(N)2 Nucleophilic Substitution Reaction.

The quaternary ammonium compound (i.e., L-carnitine for groups B11-B16or acetyl-L-carnitine for groups B21-B26) is dissolved in the polaraprotic solvent (i.e., ACN) to form the sample solution (2 μM). Thefluorescent compound (i.e., Br-MBP) is dissolved in the polar aproticsolvent (i.e., ACN) to form the fluorescent compound solution (15 mM).The sample solution is mixed with the fluorescent compound solution toform the reaction solution in accordance with the volumes indicated inTABLE 2. The reaction solution listed in TABLE 2 is incubated at 80-120°C. for 1-15 minutes to obtain the derivation solution. The derivative inthe derivation solution is extracted by the extractant (i.e., deionizedwater) in the presence of the cosolvent (i.e., toluene), the aqueousphase is then collected and mixed with 9-aminoacridine as the internalcontrol to obtain the analytic solution. Finally, the derivative in theanalytic solution is detected by the narrow-bore LC-FLD system. The peakarea ratio shown in FIG. 3 is calculated as the peak area of thederivative extracted by the extractant in the presence of the cosolventdivided by the peak area of the internal control.

TABLE 2 Reaction solution Quaternary ammonium compound (dissolvedFluorescent compound Group in ACN) (dissolved in ACN) B11 L-carnitine(20 μL) Br-MBP (3 μL) B12 Br-MBP (5 μL) B13 Br-MBP (7 μL) B14 Br-MBP (9μL) B15 Br-MBP (11 μL) B16 Br-MBP (13 μL) B21 Acetyl-L-carnitine (20 μL)Br-MBP (15 mM, 3 μL) B22 Br-MBP (15 mM, 5 μL) B23 Br-MBP (15 mM, 7 μL)B24 Br-MBP (15 mM, 9 μL) B25 Br-MBP (15 mM, 11 μL) B26 Br-MBP (15 mM, 13μL)

Referring to FIG. 3, in all of groups B11-B16 and B21-B26, the S_(N)2nucleophilic substitution reaction occurs, and the derivative can bedetected by the narrow-bore LC-FLD system. Moreover, groups B12 and B22(with 5 μL of Br-MBP dissolved in ACN) have the highest efficiency ofthe S_(N)2 nucleophilic substitution reaction, indicating that 5 μL ofBr-MBP (15 mM, dissolved in ACN) provides enough fluorescent compound toreact with the quaternary ammonium compound. As the volume of thefluorescent compound (i.e., Br-MBP dissolved in ACN) increases, thetotal volume of the reaction solution also increases; and therefore, thereactivity of the S_(N)2 nucleophilic reaction between the quaternaryammonium compound and the fluorescent compound decreases.

Trial (C): The Effect of Different Base on the S_(N)2 NucleophilicSubstitution Reaction.

The quaternary ammonium compound (i.e., L-carnitine for groups C10-C13or acetyl-L-carnitine for groups C20-C23) is dissolved in the polaraprotic solvent (i.e., ACN) to form the sample solution (2 μM). Thefluorescent compound (i.e., Br-MBP) is dissolved in the polar aproticsolvent (i.e., ACN) to form the fluorescent compound solution (15 mM).The base (i.e., KOH for groups C11 and C21, K₂CO₃ for groups C12 andC22, or KHCO₃ for groups C13 and C23) is dissolved in the polar aproticsolvent (i.e., ACN) to form the saturated basic solution. The samplesolution is mixed with the fluorescent compound solution, followed bymixing with the saturated basic solution in accordance with the volumesindicated TABLE 3. The reaction solution listed in TABLE 3 is incubatedat 80-120° C. for 1-15 minutes to obtain the derivation solution. Thederivative in the derivation solution is extracted by the extractant(i.e., deionized water) in the presence of the cosolvent (i.e.,toluene), the aqueous phase is then collected and mixed with9-aminoacridine as the internal standard to obtain the analyticsolution. Finally, the derivative in the analytic solution is detectedby the narrow-bore LC-FLD system. The peak area ratio shown in FIG. 4 iscalculated as the peak area of the derivative extracted by theextractant in the presence of the cosolvent divided by the peak area ofthe internal standard.

TABLE 3 Reaction solution Quaternary ammonium Fluorescent Group compoundcompound Base C10 L-carnitine (20 μL) Br-MBP (5 μL) None C11 KOH (2 μL)C12 K₂CO₃ (2 μL) C13 KHCO₃ (2 μL) C20 Acetyl-L-carnitine Br-MBP (5 μL)None C21 (20 μL) KOH (2 μL) C22 K₂CO₃ (2 μL) C23 KHCO₃ (2 μL)

Referring to FIG. 4, in all of groups C10-C13 and C20-C23, the S_(N)2nucleophilic substitution reaction occurs, and the derivative can bedetected by the narrow-bore LC-FLD system. Moreover, groups C11 and C21have the highest reactivity of the S_(N)2 nucleophilic substitutionreaction, indicating potassium hydroxide (KOH) provides a proper basicenvironment to facilitate the occurrence of the S_(N)2 nucleophilicsubstitution reaction, improving the reactivity of the S_(N)2nucleophilic substitution reaction.

Trial (D): The Effect of the Temperature and Time on the S_(N)2Nucleophilic Substitution Reaction.

The reaction solutions of groups C11 and C21 are incubated at 80° C.(shown in FIG. 5), 90° C. (shown in FIG. 6) or 100° C. (shown in FIG. 7)for 3, 5, 7, 9, 11 or 13 minutes to obtain the derivation solution. Thederivative in the derivation solution is extracted by the extractant(i.e., deionized water) in the presence of the cosolvent (i.e.,toluene), the aqueous phase is then collected and mixed with9-aminoacridine as the internal control to obtain the analytic solution.Finally, the analytic solution is analyzed by the narrow-bore LC-FLDsystem. The peak area ratio shown in FIGS. 5-7 is calculated as the peakarea of the derivative extracted by deionized water in the presence oftoluene divided by the peak area of the internal control.

Referring to FIGS. 5-7, in all of groups C11 and C21 at all of thetested temperature (80° C., 90° C. and 100° C.), the S_(N)2 nucleophilicsubstitution reaction occurs, and the derivative can be detected by thenarrow-bore LC-FLD system. Moreover, the S_(N)2 nucleophilicsubstitution reaction has a highest efficiency when occurring at 80° C.for more than 9 minutes. The S_(N)2 nucleophilic substitution reactionhas a highest efficiency when the S_(N)2 nucleophilic substitutionreaction has a highest efficiency when occurring at 90-100° C. for morethan 7 minutes.

Trial (E): The Effect of the Volume of the Cosolvent on the Extraction.

The reaction solution of group C11 or C21 is incubated at 80-120° C. for1-15 minutes to obtain the derivation solution. The derivative in thederivation solution is extracted by the extractant (i.e., deionizedwater) in the presence of the cosolvent (i.e., toluene), in accordancewith the volumes indicated in TABLE 4. The aqueous phase is thencollected and mixed with 9-aminoacridine as the internal control toobtain the analytic solution. Finally, the derivative in the analyticsolution is detected by the narrow-bore LC-FLD system. The peak arearatio shown in FIG. 8 is calculated as the peak area of the derivativeextracted by the extractant in the presence of the cosolvent divided bythe peak area of the internal control.

TABLE 4 Reaction Group solution Extractant Cosolvent E11 L-carnitine +Water (5 μL) Toluene (55 μL) E12 Br-MBP + KOH Toluene (65 μL) E13 (27μL) Toluene (75 μL) E14 Toluene (85 μL) E15 Toluene (95 μL) E16 Toluene(105 μL) E21 acetyl-L-carnitine + Water (5 μL) Toluene (55 μL) E22Br-MBP + Toluene (65 μL) E23 KOH (27 μL) Toluene (75 μL) E24 Toluene (85μL) E25 Toluene (95 μL) E26 Toluene (105 μL)

Referring to FIG. 8, in all of groups E11-E16 and E21-E26, the S_(N)2nucleophilic substitution reaction occurs, and the derivative can bedetected by the narrow-bore LC-FLD system. Moreover, in groups E11-E14and E21-E24, as the volume of toluene increases, the efficiency of theextraction increases because the extractant can effectively distributein the mixture containing the reaction solution, the extractant (i.e.,deionized water) and the cosolvent (i.e., toluene). However, even thevolume of the cosolvent (i.e., toluene) is over 85 μL, the efficiency ofthe extraction increases no more. That is, 27 μL of the reactionsolution is preferably mixed with 5 μL of the extractant (i.e.,deionized water) and the cosolvent (i.e., toluene).

Trial (F): The Effect of the Amount of the Extractant on the Extraction.

The reaction solution of group C11 or C21 is incubated at 80-120° C. for1-15 minutes to obtain the derivation solution. The derivative in thederivation solution is extracted by the extractant (i.e., deionizedwater) in the presence of the cosolvent (i.e., toluene), in accordancewith the volumes indicated in TABLE 5. The aqueous phase is thencollected and mixed with 9-aminoacridine as the internal control toobtain the analytic solution. Finally, the derivative in the analyticsolution is detected by the narrow-bore LC-FLD system. The peak arearatio shown in FIG. 9 is calculated as the peak area of the derivativeextracted by the extractant in the presence of the cosolvent divided bythe peak area of the internal control.

TABLE 5 Reaction Group solution Extractant Cosolvent F11 L-carnitine +Water (5 μL) Toluene (85 μL) F12 Br-MBP + KOH Water (7 μL) F13 (27 μL)Water (9 μL) F14 Water (11 μL) F15 Water (13 μL) F16 Water (15 μL) F21acetyl- Water (5 μL) Toluene (85 μL) F22 L-carnitine + Water (7 μL) F23Br-MBP + KOH Water (9 μL) F24 (27 μL) Water (11 μL) F25 Water (13 μL)F26 Water (15 μL)

Referring to FIG. 9, in all of groups F11-F16 and F21-F26, thederivative can be extracted and then be detected by the narrow-boreLC-FLD system. As the volume of deionized water decreases, the peak arearatio increases. However, the aqueous phase is difficult to be collectedif the volume of the extractant is below 5 μL. Therefore, 27 μL of thereaction solution is preferably mixed with 5 μL of the extractant (i.e.,deionized water) and the cosolvent (i.e., toluene).

Trial (G): The Effect of Different Extractant on the Extraction.

The reaction solution of group C11 or C21 is incubated at 80-120° C. for1-15 minutes to obtain the derivation solution. The derivative in thederivation solution is extracted by the extractant (i.e., deionizedwater for groups G10 and G20, NH₄OAC_((aq)) for groups G11 and G21,NH₄Cl_((aq)) for groups G12 and G22, NaCl_((aq)) for groups G13 and G23,or NH₄HCO_(3(aq)) for groups G14 and G24) in the presence of thecosolvent (i.e., toluene), in accordance with the volumes indicated inTABLE 6. The aqueous phase is then collected and mixed with9-aminoacridine as the internal control to obtain the analytic solution.Finally, the derivative in the analytic solution is detected by thenarrow-bore LC-FLD system. The peak area ratio shown in FIG. 10 iscalculated as the peak area of the derivative extracted by theextractant in the presence of the cosolvent divided by the peak area ofthe internal control.

TABLE 6 Reaction Group solution Extractant Cosolvent G10 L-carnitine +Water (5 μL) Toluene (85 μL) G11 Br-MBP + KOH NH₄OAc_((aq)) (27 μL) (1M,5 μL) G12 NH₄Cl_((aq)) (1M, 5 μL) G13 NaCl_((aq)) (1M, 5 μL) G14NH₄HCO_(3(aq)) (1M, 5 μL) G20 acetyl-L-carnitine + Water (5 μL) Toluene(85 μL) G21 Br-MBP + NH₄OAc_((aq)) KOH (27 μL) (1M, 5 μL) G22NH₄Cl_((aq)) (1M, 5 μL) G23 NaCl_((aq)) (1M, 5 μL) G24 NH₄HCO_(3(aq))(1M, 5 μL)

Referring to FIG. 10, in all of groups G10-G14 and G20-G24, thederivative can be extracted and then be detected by the narrow-boreLC-FLD system. Moreover, deionized water in group G10 or G20 shows thehighest peak area ratio, followed by NH₄OAc_((aq)) in group G11 or G21.

Trial (H): The Effect of Different Extractant on the Extraction of theDerivative in the Plasma Sample.

Human plasma is mixed with a protein precipitation reagent to remove theprotein in said human plasma, and then dissolved in ACN, followed bymixing with the quaternary ammonium compound (i.e., L-carnitine forgroups H10-H14, acetyl-L-carnitine for groups H20-H24 or butyrobetainefor groups H30-H34) and the fluorescent compound (i.e., Br-MBP) to formthe reaction solution. The reaction solution is incubated at 80-120° C.for 1-15 minutes to obtain the derivation solution. The derivative inthe derivation solution is extracted by the extractant (i.e., deionizedwater for groups H10, H20 and H30, NH₄OAC_((aq)) for groups H11, H21 andH31, NH₄Cl_((aq)) for groups H12, H22 and H32, NaCl_((aq)) for groupsH13, H23 and H33, or NH₄HCO_(3(aq)) for groups H14, H24 and H34) in thepresence of the cosolvent (i.e., toluene), in accordance with TABLE 7.The aqueous phase is then collected and mixed with 9-aminoacridine asthe internal control to obtain the analytic solution. Finally, thederivative in the analytic solution is detected by the narrow-boreLC-FLD system. The peak area ratio shown in FIG. 11 is calculated as thepeak area of the derivative extracted by the extractant in the presenceof the cosolvent divided by the peak area of the internal control.

TABLE 7 Reaction solution Quaternary ammonium Fluorescent Group compoundcompound Extractant Cosolvent H10 L-carnitine Br-MBP Water Toluene H11NH₄OAc_((aq)) (1M) H12 NH₄Cl_((aq)) (1M) H13 NaCl_((aq)) (1M) H14NH₄HCO_(3(aq)) (1M) H20 Acetyl-L-carnitine Br-MBP Water Toluene H21NH₄OAc_((aq)) (1M) H22 NH₄Cl_((aq)) (1M) H23 NaCl_((aq)) (1M) H24NH₄HCO_(3(aq)) (1M) H30 Butyrobetaine Br-MBP Water Toluene H31NH₄OAc_((aq)) (1M) H32 NH₄Cl_((aq)) (1M) H33 NaCl_((aq)) (1M) H34NH₄HCO_(3(aq)) (1M)

Referring to FIG. 11, in all of groups H10-H14, H20-H24 and H30-H34, thederivative can be extracted and then be detected by the narrow-boreLC-FLD system. Moreover, for the plasma sample, NH₄OAc_((aq)) in groupH11, H21 or H31 shows the highest peak area ratio, followed byNH₄Cl_((aq)) in group H12, H22 or H32.

Trial (I): Analysis of the Plasma Sample by the Nano LC-MS/MS System.

Human plasma (1 μL) containing difference quaternary ammonium compoundin accordance with TABLE 8 is pretreated as mentioned above. The formedreaction solution is incubated at 80-120° C. for 1-15 minutes to obtainthe derivation solution. The derivative in the derivation solution isextracted by the extractant (i.e., deionized water), the aqueous phaseis then collected to obtain the analytic solution. Finally, thederivative in the analytic solution is detected by the nano LC-MS/MSsystem.

TABLE 8 Quaternary ammonium Formula Molar mass Measured Error compound[M]⁺ (Da) mass (Da) (ppm) MS²(m/z) Butyrobetaine C₂₀H₂₆NO₂ 312.1958312.1955 −0.96 152, 167, 226 L-carnitine C₂₀H₂₆NO₃ 328.1907 328.1908−0.30 152, 167, 226, 310 Acetyl-L- C₂₂H₂₈NO₄ 370.2012 370.2008 −1.08152, 167, carnitine 226 Propionyl- C₂₃H₃₀NO₄ 384.2175 384.2165 −2.60152, 167, L-carnitine 226, 366 Butyryl-L- C₂₄H₃₂NO₄ 398.2331 398.2325−1.50 152, 167, carnitine 226, 380 Isovaleryl- C₂₅H₃₄NO₄ 412.2487412.2479 −1.94 152, 167, L-carnitine 226, 394 Hexanoyl- C₂₆H₃₆NO₄426.2644 426.2638 −1.41 152, 167, L-carnitine 226, 408 Octanoyl-C₂₈H₄₀NO₄ 454.2957 454.2954 −0.66 152, 167, L-carnitine 226, 436Decanoyl- C₃₀H₄₄NO₄ 482.3270 482.3265 −1.03 152, 167, L-carnitine 226,464 Lauroyl-L- C₃₂H₄₈NO₄ 510.3583 510.3559 −4.70 152, 167, carnitine226, 492 Myristoyl- C₃₄H₅₂NO₄ 538.3891 538.3884 −1.30 152, 167,L-carnitine 226, 520 Palmitoyl- C₃₆H₅₆NO₄ 566.4204 566.4203 −0.18 152,167, L-carnitine 226, 548 Stearoyl-L- C₃₈H₆₀NO₄ 594.4517 594.4516 −0.17152, 167, carnitine 226, 576

Referring to TABLE 8, the detection method for the quaternary ammoniumcompound having the γ-carboxyl group can be used to detect more than 13carnitine derivatives in the human body by the nano LC-MS/MS system.

Trial (J): Analysis of Commercial Tablets.

The tablet containing L-carnitine is ground, dissolved in ACN andcentrifuged to collect a supernatant used in subsequent S_(N)2nucleophilic substitution and extraction. The amount of L-carnitinedetected by the liquid chromatography is listed in TABLE 9. The claimedamount of the tablet is 1 gram L-carnitine per tablet.

TABLE 9 Found amount No. (mg/tablet) RSD (%) Recovery (%) #1 964.94 1.8796.49 #2 1002.33 3.95 100.23 #3 1023.30 3.07 102.33 #4 972.73 5.10 97.27#5 995.68 3.01 99.57

Referring to TABLE 9, the detection method for the quaternary ammoniumcompound having the γ-carboxyl group can be used to detect theL-carnitine amount in the pharmaceuticals in tablet formulation.Accordingly, based on the result of the detection method for thequaternary ammonium compound having the γ-carboxyl group, a personhaving ordinary skill in the art can determine whether the analyzedpharmaceutical sample corresponds with the labeling regulation. As anexample, to correspond with United States Pharmacopeia (USP), the foundamount of the tablet formulation containing L-carnitine should be rangedbetween 90.0% and 110.0% of the claimed amount.

Trial (K): Analysis of Commercial Injections.

The injection containing L-carnitine is dissolved in ACN and centrifugedto collect a supernatant used in subsequent S_(N)2 nucleophilicsubstitution and extraction. The amount of L-carnitine detected by theliquid chromatography is listed in TABLE 10. The claimed amount of theinjection is 1 gram L-carnitine per 5 mL injection.

TABLE 10 Found amount No. (mg/mL) RSD (%) Recovery (%) #1 204.15 3.91102.08 #2 208.75 2.24 104.38 #3 202.56 5.43 101.28 #4 203.48 2.40 101.74#5 202.21 5.09 101.11

Referring to TABLE 10, the detection method for the quaternary ammoniumcompound having the γ-carboxyl group can be used to detect L-carnitineamount in the pharmaceuticals in injection formulation. Accordingly,based on the result of the detection method for the quaternary ammoniumcompound having the γ-carboxyl group, a person having ordinary skill inthe art can also determine whether the analyzed pharmaceutical samplecorresponds with the labeling regulation. As an example, to correspondwith United States Pharmacopeia (USP), the found amount of the injectionformulation containing L-carnitine should be ranged between 90.0% and110.0% of the claimed amount.

Trial (L): Analysis of Commercial Food Items and Cosmetics.

The food item containing L-carnitine (No. #01-#04) or the cosmeticscontaining L-carnitine or (No. #05-#09) or acetyl-L-carnitine (No.#10-#12) is dissolved in ACN and centrifuged to collect a supernatantused in subsequent S_(N)2 nucleophilic substitution and extraction. Theamount of L-carnitine (or acetyl-L-carnitine) detected by the liquidchromatography is listed in TABLE 11. The claimed amounts of the fooditems #01, #02 and #03 are 500 mg/g, 250 mg/g and 20 mg/250 mL,respectively.

TABLE 11 Found amount Recovery No. L-carnitine acetyl-L-carnitine RSD(%) (%) #01 441.55 mg/g — 7.87 88.3 #02 246.94 mg/g — 2.91 98.9 #0387.72 μg/g — 1.93 109.7 #04 0.66 μg/g — 1.53 — #05 2885.25 μg/g — 6.68 —#06 0.89 μg/g — 1.44 — #07 330.61 μg/g — 3.23 — #08 15280.90 μg/g — 1.13— #09 489.23 μg/g — 3.28 — #10 — 492.84 μg/g 3.56 — #11 — 480.89 μg/g0.52 — #12 — 166.69 μg/g 1.65 —

Referring to TABLE 11, the detection method for the quaternary ammoniumcompound having the γ-carboxyl group can be used to detect L-carnitineamounts in various sample types, such as food items, cosmetics etc.

In view of the above experiment results, by the addition of thefluorescent compound and the polar aprotic solvent, the quaternaryammonium compound can be labeled by the fluorophore via the S_(N)2nucleophilic substitution reaction with the fluorescent compound. Thefluorophore-labeled quaternary ammonium compound can therefore beanalyzed by the fluorescence spectrometry. Moreover, compared to thequaternary ammonium compound, the fluorophore-labeled quaternaryammonium compound has a lower polarity, as well as a longer retentiontime when being analyzed by reverse-phase HPLC.

Moreover, the fluorophore-labeled quaternary ammonium compound isconcentrated by the extractant, and the sensibility to detect thequaternary ammonium compound is therefore improved.

In addition, the diagnostic method for carnitine deficiency can be usedto effectively tell whether the suspected patient suffers from carnitinedeficiency by precisely measuring the L-carnitine amount in the sampleobtained from the suspected patient.

Furthermore, by using the detection kit for the quaternary ammoniumcompound, the worker can convert the quaternary ammonium compound in thesample into the fluorophore-labeled quaternary ammonium compound (i.e.,the derivative) via the S_(N)2 nucleophilic substitution reaction. Thederivative can be subjected to subsequent analysis, such as liquidchromatography, fluorescence spectrometry or mass spectrometry.

Although the invention has been described in detail with reference toits presently preferable embodiment, it will be understood by one ofordinary skill in the art that various modifications can be made withoutdeparting from the spirit and the scope of the invention, as set forthin the appended claims.

What is claimed is:
 1. A detection kit for the quaternary ammoniumcompound having a γ-carboxyl group, comprising: a fluorescent compoundhaving a fluorophore, wherein the fluorophore of the fluorescentcompound is used for forming a derivative with a γ-carboxyl group of thequaternary ammonium compound by an S_(N)2 nucleophilic substitutionreaction; and a polar aprotic solvent used for accelerating the S_(N)2nucleophilic substitution reaction.
 2. The detection kit for thequaternary ammonium compound having the γ-carboxyl group as claimed inclaim 1, wherein the fluorescent compound having the fluorophore isselected from 4-bromomethylbiphenyl, 4-bromoethylbiphenyl or4-bromomethyl-2′-cyanobiphenyl.
 3. The detection kit for the quaternaryammonium compound having the γ-carboxyl group as claimed in claim 1,wherein the polar aprotic solvent is acetonitrile.
 4. The detection kitfor the quaternary ammonium compound having the γ-carboxyl group asclaimed in claim 1, wherein the detection kit comprises: a base being abasic compound able to dissolve in the polar aprotic solvent and beingused for providing a basic environment for performing the S_(N)2nucleophilic substitution reaction.
 5. The detection kit for thequaternary ammonium compound having the γ-carboxyl group as claimed inclaim 4, wherein the base is selected from potassium hydroxide,potassium carbonate or potassium bicarbonate.
 6. The detection kit forthe quaternary ammonium compound having the γ-carboxyl group as claimedin claim 1, wherein the detection kit comprises: an extractant having apolarity higher than a polarity of the polar aprotic solvent and beingused for extracting the derivative.
 7. The detection kit for thequaternary ammonium compound having the γ-carboxyl group as claimed inclaim 6, wherein the detection kit comprises: a cosolvent used forforming a water-in-oil emulsion with the extractant.
 8. The detectionkit for the quaternary ammonium compound having the γ-carboxyl group asclaimed in claim 7, wherein the extractant is selected from deionizedwater, an aqueous ammonium acetate solution, an aqueous ammoniumchloride solution, an aqueous sodium chloride solution or an aqueousammonium bicarbonate solution, wherein the cosolvent is toluene.